MeCP2 (methyl-CpG binding protein 2) functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Several lines of evidence exist to support the possibility that differential phosphorylation of MeCP2 in response to neuronal activity may serve as a molecular switch in dynamically modulating neuronal gene expression, which underlies the activity-dependent phase of mammalian brain development. First, MeCP2 expression is dramatically up-regulated in mature neurons during the period of synaptic refinement. Second, Rett Syndrome (RTT, an autism spectrum developmental disorder caused by mutations in the MECP2 gene) patients are born normal (suggesting MeCP2 is not required for activity-independent embryonic brain development), but become symptomatic during the period of synaptic refinement (suggesting MeCP2 is required for activity-dependent postnatal brain development). Third, two in vitro studies showed that neuronal activity-induced phosphorylation at serine 421 (S421) precedes the release of MeCP2 from the neuronal specific promoter of the brain-derived neurotrophic factor (BDNF) gene and the subsequent expression of BDNF. Finally, comprehensive biochemical analysis has identified 8 phosphorylation sites on the MeCP2 protein. Among these, serine 80 (S80) is phosphorylated in resting neurons but dephosphorylated in active neurons, whereas S421 is dephosphorylated in resting neurons but phosphorylated in active neurons. To determine how neuronal activity induced differential phosphorylation of MeCP2 fine-tunes the promoter occupancy of MeCP2 across the genome and induces corresponding changes in chromatin marks, we have generated several novel Mecp2 knock-in alleles carrying point mutations that either abolish or mimic phosphorylation at S80 and S421 on the MeCP2 protein, as well as a FLAG tag at the carboxyl terminal of the MeCP2 protein. As a part of our long-term goal to understand the dynamic role of MeCP2 in DNA methylation-dependent epigenetic regulation of mammalian brain development and functions, we propose to: 1) perform ChIP-chip (chromatin immunoprecipitation followed by hybridization onto a DNA oligo array) experiments to reveal how changes in the phosphorylation status of MeCP2 cause changes in its ability to bind to gene promoters across the entire genome;2) perform ChIP-chip experiments to reveal how changes in the phosphorylation status of MeCP2 induce corresponding changes in chromatin marks at its target gene promoters across the entire genome. PUBLIC HEALTH RELEVANCE: Mutations in the X-linked human MECP2 gene (methyl-CpG binding protein 2) cause Rett syndrome (RTT), an autism spectrum developmental disorder that predominantly affects females. To understand the molecular mechanism of RTT, it is important to study how MeCP2 dynamically regulates gene transcription. Results from this study will advance our understanding of the molecular mechanism of Rett syndrome (RTT). Furthermore, because of the considerable overlap in clinical features between RTT and autistic spectrum disorders, the lessons learned studying RTT might also benefit the general understanding of autism.